The long-term objective of the proposed research is to understand the cellular mechanisms for the repair of DMA double-strand breaks (DSBs) in eukaryotes, using budding yeast as a model system. The ability to repair DSBs by HR is essential for cell proliferation in vertebrates and for genome stability in all characterized organisms. Central to this process is the Rad51 complex with ssDNA, which is active in the homology search and strand exchange phases of the reaction. However, unregulated recombination is likely to be detrimental to cells and the Rad51 filament appears to be a common target for regulation by a number of proteins. Rad52, Rad54, Rad55 and Rad57 function in formation and/or stabilization of the Rad51 nucleoprotein filament, whereas Srs2 functions to destabilize the filament. Brca2 appears to have complex roles in the regulation of Rad51 by promoting nuclear entry, targeting Rad51 to sites of DMA damage and destabilization of the Rad51 filament. Because of the central role of Rad51 filament dynamics for appropriate recombination in response to DMA damage, it is important to have a complete understanding of the formation and disassembly of the Rad51/DNA complex. The first aim of this proposal builds on our previous studies of rad51 gain-of-function and altered function alleles to further understand how the Rad51 filament interacts with Rad55, Rad57 and Srs2. The function of residues identified at the protomer interface in the crystal structure of the Rad51 filament will also be determined. The second aim of the proposal is to characterize known suppressors, and identify additional suppressors of rad57, to determine whether Rad57 functions exclusively at the level of the Rad51 filament, or has additional late roles in recombination. The roles of Rad55 and Rad57 in spontaneous and induced, intra and inter-chromosomal recombination will also be determined. The last aim includes in vitro studies to identify preferred DNA substrates for Rad55:Rad57 binding and stabilization of the Rad51 nucleoprotein by Rad55:Rad57 in the presence or absence of Srs2.